We wish to define the role of reversible protein methylation in eucaryotic cells, particularly in human blood cells. We have shown that specific erythrocyte cytoskeletal and membrane proteins are methylated and demethylated in ester linkages at aspartyl residues. We are now concerned with the question of the physiological consequences of these reaction. Is the shape and deformability of the red cell, or its capacities to transporat anions and other metabolites across the plasma membrane, regulated by the level of methylation of these proteins? To pursue these questions we will ask whether the methylation of membrane proteins separated by dodecyl sufate gel electrophoresis from intact cells labeled with [3H-methyl] methionine is affected by the various conditions that a red cell is normally exposed to in the curculation. These conditions include plasma hormone levels as well as mechanical stresses on the membrane as erythrocytes pass through the capillary beds. We can also ask whether changes in protein methylation (induced for example by incubating cells with permeable inhibitors of the cytosolic protein methyltransferase) can affect any of the functional properties of red cells, or whether the in vitro activities of purified membrane proteins are modified by their degree of methylation. We will be interested in the factors that control the in vivo state of methylation and will pursue the identification and characterization of the protein carboxyl methyltransferase(s), protein methylestera(s), and endogenous small molecule substrates and inhibitors. We also propose to characterize protein methylation reactions in other types of cells. We will compare the methylation of membrane proteins in normal human erythrocytes with those of patients with disease expressed in these cells such as hereditary sperocytosis, sickle cell anemia, and muscular dystrophy. These experiments may not only provide evidence of the function of methylation in normal cells but may shed insight on the molecular nature of these disease. Because protein carboxyl methylation has already been shown to be involved in receptor function in bacterial chemotaxis, we will also investigate whether reversible methylation reactions play a role in human platelet functions such as clot retraction and colagen-and thrombin-induced shape changes and aggregation or in human neutrophil functions such as chemotaxis. Rats, rabbits, cats, dogs, chicken, sheep.